BG - 581
DESENVOLVIMENTO: A HISTORIA NATURAL DOS GENES
Paulo Arruda
10-03-2011
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO
Matthew P. Scott (2000) Development: The Natural History of Genes. Cell, 100: 27-40
(1) Tipos celulares diferentes transcrevem genes diferentes. Celulas citologicamente
indistinguíveis podem ter padrões elaborados de expressão genica temporal e espacial.
(2) Embora alterações no DNA são utilizadas para determinar a ação diferencial dos genes,
o controle da transcrição é a forma mais comum de regulação da expressão gênica.
(3) A explosão transiente da ação de fatores de transcrição ou sinalização leva a alterações
permanentes no fenótipo celular, que embora, muitas vezes reversíveis, podem
perpetuar decisões regulatórias precoces.
(4) Moléculas de RNA localizadas criam diferenças no citoplasma do óvulo ou entre celulas,
criando divisões celulares assimétricas. Divisão celular assimétrica é crucial para a
diferenciação de células tronco.
(5) Fatores de transcrição regulam baterias de genes. Combinações de reguladores
governam a maioria dos eventos de diferenciação. O resultado depende da ação em
concerto ou em oposição de reguladores.
(6) Os embriões são flexíveis em se adaptar ao número de células, a condições ambientais
e alterações na ação gênica. As células são sensíveis à qualidade das células vizinhas e
tendem a morrer ou mudar se as células vizinhas não são apropriadas. Elas são tambem
sensíveis ao número de células vizinhas “efeito da comunidade”.
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO
(7) Muitos sinais agem a curta distancia, talvez refletindo suasw origens no organismo com
poucas células.
(8) Sistemas de checagem controlam a açõa de sinalizadores. Antagonistas intra- e extracelulares limitam a atuação de sinalizadores sendo que alguns são induzidos pelos seus
sinalizadores proporcionam um mecanismo de retroalimentação.
(9) As células são responsivas a sinalizadores ou fatores de transcrição em algumas
condições e não em outras, criando a propriedade denominada de competencia. As
células respondem diferencialmente a um sinalizador particular, dependendo da sua
história. A historia pode afetar a efetividade, tornando certos genes responsívos a
sinalizadores atraves da localização de moléculas dentro das células.
(10) A regulação temporal e espacial do ciclo celular é crucial para a formação de tecidos
organizados,permitindo a restauração e manut6enção das células tronco.
(11) A morte celular e o envelhecimento são processsos governados geneticamente atraves
de “scripts” precisos durante o desenvolvimento normal e alterados em condições de
doença e injúria.
(12) A maioria das proteínas que regulam o desenvolvimento foram conservadas durante a
evolução. Muitas vezes, genes conservados tem funções imilares numa vasta gama de
diferentes animais.
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO
Expressão espacial altamente precisa
do gene patched1 regulado por
Hedgehog. Hedgehog indus a
expressão de patched1 e outros genes
alvo num padrão espacial e temporal
preciso, mostrado aqui pela inserção
do gene lacZ no locus patched1. A
regulação da diferenciação por
Hedgehog é amplamente conservada
em animais.
Laboratório de Análise
da Regulação da
Expressão Gênica
DEVELOPMENT OF EYE COLORS IN DROSOPHILA: SOME
PROPERTIES OF THE HORIVIONES CONCERNED
Tatum EL and Beadle GW (1938) The journal of general physiology. X: 239-253
Abstract
Diffusible substances showing hormone-like action in modifying the development
of eye color are known in Drosophila and in certain other insects. These
substances are active when injected into, or fed to, appropriate test larvae. The so
called v+ substance causes a modification of the eye color of vermilion brown (v
bw) animals from a pale pink towards brown. Similarly, cn+ substance induces a
modification of cinnabar brown (cn bw) eye color from colorless towards brown.
This paper reports some of the further information which has been obtained
primarily in regard to the properties, chemical nature, and possible methods of
purification of the v+ hormone.
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO
Método:
The v+ hormone used for this work was obtained from 24-48 hour old wild type
pupae. The pupae were washed and dried at 100°C. for 1 hour. This served both
to inactivate the enzymes and to dry the material. The dried pupae were kept
under reduced pressure until needed. The hormone was extracted from finely
ground dried pupae by procedures to be described later in this paper. Small
aUquots of the extracts to be tested were dried under reduced pressure at 100°C
weighed, and dissolved in Ringer's solution and, if necessary, the pH adjusted to
6.0. The solutions prepared in this way were sealed in small glass tubes and
immersed in boiling water for 10 minutes. This was found to be necessary to
prevent bacterial growth and consequent toxicity for the test animals. Solutions
prepared in this way retained their activity indefinitely. The activity of the various
solutions was determined by injecting them into the body cavities of v bw larvae
shortly before puparium formation. The effect manifested itself in a modification of
the eye color from neatly colorless towards brown. Ten to fifteen larvae were
injected with each solution to be tested,
Laboratório de Análise
da Regulação da
Expressão Gênica
EFEITO DO HORMONIO v+ NA COR DO OLHO DE MOSCAS v bw
Laboratório de Análise
da Regulação da
Expressão Gênica
EXTRAÇÃO DO HORMONIO v+ DAS PUPAS
The ground dried pupae were extracted several times with boiling chloroform, and
this inactive extract was discarded. The hormone was then extracted by repeated
treatments with hot 95 per cent ethyl alcohol. The alcohol was removed from the
yellow extract under reduced pressure and the residue taken up in distilled water.
The water-insoluble portion of this mixture was removed by centrifuging. The final
product was a fairly clear yellow solution. The results of a number of extractions
are summarized in Table II. Approximately 5 per cent of the original weight of the
pupae were contained in the final extract. Disregarding a few inexplicable cases in
which extracts of quite low activity were obtained, the average activity was about
280,000 units per gram extract. This means that from 1 gm. of dried pupae around
17,500 units were extracted.
Laboratório de Análise
da Regulação da
Expressão Gênica
EXTRAÇÃO DO HORMONIO v+ DAS PUPAS
Laboratório de Análise
da Regulação da
Expressão Gênica
PROPRIEDADES DO HORMONIO v+
Laboratório de Análise
da Regulação da
Expressão Gênica
INATIVAÇÃO ENZIMÁTICA DO HORMONIO v+
Since the hormone had been shown to be relatively stable towards oxidation, it
was thought advisable to investigate its inactivation by the enzymes present in the
larvae and pupae. Juice obtained by crushing or grinding fresh pupae or larvae
and filtering, was mixed with concentrated active extract. The solutions were
allowed to stand for 15-20 hours on watch glasses at 30°C., then diluted with
Ringer's solution to known volumes, sealed in glass tubes, heated, centrifuged,
and tested by injection.
Laboratório de Análise
da Regulação da
Expressão Gênica
INATIVAÇÃO ENZIMÁTICA DO HORMONIO v+
In no case using juice from
crushed pupae or larvae did
inactivation result. However,
the juice from wild type or
vermilion larvae and wild
type pupae obtained by
grinding the material
thoroughly with finely
powdered silica, almost
completely inactivated the v+
hormone
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE É O HORMONIO v+?
Although all the data indicate the amino acid nature of the hormone, it seems
probable that it is not a simple amino acid. The substance may be destroyed or
inactivated by many treatments which would not affect simple amino acids. The
instabillty of the hormone to heat, and to treatment with acid and alkali illustrate
this point. Furthermore, the high molecular weight of 400 to 600 as determined by
the diffusion method also eliminates the common simple amino acids from
consideration. the exact chemical nature of the hormone. However, there seem to
be three distinct possibilities. It may prove to be a complex single amino acid, a
peptide of several amino acids, or it may be a compound of one or more amino
acids with other substances. In any case it is probably not a normal constituent of
tissue, since its occurrence seems to be rather strictly limited to certain insects.
Khouvine and Ephrussi were unable to demonstrate any hormone activity in
nitrogenous extracts of sheep brain, while similarly prepared extracts of CaP
liphora were very active. Although yeast is generally supposed to contain most
known nitrogenous substances, we have had no indication that it contains this
particular amino acid-like hormone.
Laboratório de Análise
da Regulação da
Expressão Gênica
GENETIC CONTROL OF BIOCHEMICAL REACTIONS IN
NEUROSPORA
Beadle GW and Tatum EL (1941) PNAS
Summary.
-A procedure is outlined by which, using Neurospora, one can discover and
maintain x-ray induced mutant strains which are characterized by their inability to
carry out specific biochemical processes. Following this method, three mutant
strains have been established. In one of these the ability to synthesize vitamin B6
has been wholly or largely lost. In a second the ability to synthesize the thiazole
half of the vitamin B1 molecule is absent, and in the third para-aminobenzoic acid
is not synthesized. It is therefore clear that all of these substances are essential
growth factors for Neurospora-11Growth of the pyridoxinless mutant (a mutant
unable to synthesize vitamin B6) is a function of the B6 content of the medium on
which it is grown. A method is described for measuring the growth by following
linear progression of the mycelia along a horizontal tube half filled with an agar
medium. Inability to synthesize vitamin B6 is apparently differentiated by a single
gene from the ability of the organism to elaborate this essential growth substance.
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO
Laboratório de Análise
da Regulação da
Expressão Gênica
O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO
Laboratório de Análise
da Regulação da
Expressão Gênica
GENETIC CONTROL OF BIOCHEMICAL REACTIONS IN NEUROSPORA:
AN "AMINOBENZOICLESS" MUTANT
By E. L. TATUM AND G. W. BEADLE (1942) PNAS
Summary
An x-ray induced mutant strain of Neurospora crassa has been obtained which
requires p-aminobenzoic acid for growth. Its growth is a function of the amount of
p-aminobenzoic acid supplied, and it is indistinguishable from normal when
adequate amounts of p-aminobenzoic acid are available. The mutant differs from
normal by a single gene, which must therefore control an essential step in the
synthesis of p-aminobenzoic acid, and which is presumably primarily concerned
only with the synthesis of p-aminobenzoic acid. Sulfanilamide inhibits the growth of
both the normal and mutant strains, and in both cases the inhibition is overcome
by an excess of p-aminobenzoic acid. A number of substances related to paminobenzoic acid are able to replace it, but their activities are much less than that
of p-aminobenzoic acid itself. The addition of benzoic or p-OH-benzoic acids or
tyrosine did not increase the amount of p-aminobenzoic acid produced by the
normal strain. It is concluded that none of the compounds tested is concerned with
the normal synthesis of p-aminobenzoic acid, and that this biosynthesis probably
does not involve the introduction of an amino group into a preformed benzene ring.
Laboratório de Análise
da Regulação da
Expressão Gênica
THE NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE 1958
"for their discovery that genes act by
regulating definite chemical events"
"for his discoveries concerning genetic
recombination and the organization of
the genetic material of bacteria"
George Wells Beadle
Edward Lawrie Tatum
Joshua Lederberg
1/4 of the prize
1/4 of the prize
1/2 of the prize
California Institute of
Technology (Caltech)
Pasadena, CA, USA
Rockefeller Institute for
Medical Research
New York, NY, USA
University of Wisconsin
Madison, WI, USA
b. 1903
d. 1989
b. 1909
d. 1975
b. 1925
d. 2008